From January 2014 to June 2015, 86 terminal colorectal cancer patients were enrolled in this study. None of these cases had received any type of cancer treatment prior to their enrollment. The estimated survival time of the patients was ~12 months. After enrollment, the patients were treated with oxaliplatin combined with gemcitabine and all patients completed the course of treatment even those who suffered from severe side effects. Karnofsky performance status scoring was >70 points; hemoglobin level was ≥90 g/l, neutrophil differential count was ≥2.0×10⁹/l, blood platelet count was ≥100×10⁹/l; glutamic-pyruvic transaminase was within normal range and no severe organ dysfunction was reported. The patients included 50 males and 36 females aged from 48 to 73 years (average age, 57.6±13.3 years). According to Dukes staging system, 57 patients were in stage C2 and 29 cases were in stage D, while adenocarcinoma was the most common histologic type. The maximum diameter of tumors ranged from 3.0 to 6.2 cm (average, 5.3±1.4 cm). There were 66 cases with lymphatic metastasis, 13 cases with liver metastasis and 7 cases with spread of cancer to the lung and other parts of the body. This study was approved by the ethics committees of the participating hospitals, and all patients and their family members provided conformed consent.

We used the following doses of gemcitabine and oxaliplatin: the gemcitabine dose was 1,000 mg/m² ivgtt in 100 ml normal saline for 30–60 min at days 1 and 8 of the cycle; and oxaliplatin: 100 mg/m² ivgtt for 2 h at day 2 of the cycle. There were 21 days in each cycle and patients were treated for 2 cycles under close observation. For severe stenosis of bowel and adhesion, we performed minimally invasive palliative surgery for resection and colorectal colonoscopy was conducted for tissue sampling. All procedures were conducted following patient consent. The follow-up visits continued for 18 months.

The total effective rate was analyzed based on WHO tumor chemotherapy reference where complete remission (CR) implies complete disappearance of the tumor, partial remission (PR) implies that the tumor size was reduced at least by 50%, stable disease (SD) implies that the tumor size remained unchanged and progressive disease (PD) implies an increase in the size of the tumor by ≥25%. CT scan examinations were conducted to measure the size of the tumors. Immunohistochemistry was used to study the subcellular localization of HMGB1 in the cancer tissues as well as the para-carcinoma tissues (≥10 cm away from the tumor edge). RT-PCR and western blotting were used to assess the mRNAand protein expression level, respectively. Conventional method was used for tissue preparation as follows. The samples were dewaxed using xylene, washed with gradient alcohol, stained with hematoxylin, dehydrated by ethyl alcohol, and the samples were sealed by neutral resins followed by washing with phosphate-buffered saline (PBS). We added induced antigen retrieval solution and goat serum blocking reagent. The primary antibody was then added followed by washing and addition of the secondary antibody (R&D Systems, Inc., Minneapolis, MN, USA). Samples were washed with PBS and streptavidin peroxidase was added and then DAB color developing agent was added after PBS washing and counterstaining was carried out using hematoxylin. Intensity of staining (IHS) was calculated using the following formula: IHS = A × B, where ‘A’ is the number of positive cells and ‘B’ is the intensity of the color. Positive expression was considered at IHS of 3.

RT-PCR. Conventional TRIzol reagent method was used for RNA extraction and ultraviolet absorption spectroscopy method was used to verify the concentration and purity. SuperScript™ III Reverse Transcriptase kit (ABI, Invitrogen Life Technologies, Carlsbad, CA, USA) was used for cDNA synthesis and Primer 3.0 online software was used to design the primers: HMGB1 (F), 5′-ATATGGCAAAAGCGGACAAG-3′ and HMGB1 (R), 5′-AGGCCAGGATGTTCTCCTTT-3′. The internal control was β-actin (F), 5′-CTCTGGCCGTACCACTGGC-3′, and β-actin (R), 5′-GTGAAGCTGTAGCCGCGC. The reaction system used was the following: l µl of cDNA, 2 µl of 10X buffer, l µl of Mg²⁺, l µl assay and 0.2 µl Taq and 14.8 µl of ddH₂O. We used ABI 7500 qPCR apparatus and the parameters were: 95°C for 2 min, 94°C for 20 sec, 60°C for 20 sec and 72°C for 30 sec for a total of 40 cycles. Fluorescent quantitation PCR was used to monitor the expression of HMGB1 mRNA, and Ct in all samples was tested using fold = 2^−ΔΔCt to express the multiple proportion relation between the target gene and internal control gene. Samples were testing 3 times and the average values were recorded.

Western blot analysis. The HMGB1 antibody was purchased from Abcam Company, the secondary antibody was obtained from R&D Systems, Inc., and the β-actin antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Trans-blot was obtained from Bio-Rad (Berkeley, CA, USA). For imaging, we used a gel image analysis system (UVP Co., San Gabriel, CA, USA). Conventional RIPA lysate was used to extract total protein, and the BCA protein quantification kit was purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA). The concentration was assessed using a microplate reader. Proteins were separated using SDS-PAGE, and the separated proteins were transferred and blotted onto a polyvinylidene fluoride membrane. Next, the membranes were blocked and chemiluminescence was performed.

We used the Statistical Package for Social Sciences (SPSS, Inc., Chicago, IL, USA) for our statistical analyses. Data are presented as mean ± standard deviation (SD) and proportions. Continuous data are presented as mean ± SD. Categorical values were evaluated using the Chi-square test. Survival was analyzed by Kaplan-Meier method. P<0.05 was considered to indicate a statistically significant difference.

We identified 20 cases of CR, 37 cases of PR, 12 cases of SD and 17 cases of PD. The total effective rate (CR+PR) was 66.3%. Positive expression of HMGB1 protein was mainly localized in the karyon (light yellow and dark yellow particles). In the effective group, we observed 12 cases (21.1%) of positive expression of HMGB1 in the cancer tissues while in the ineffective group the number of positive cases was 12 (41.4%). HMGB1 expression in the cancer tissues obtained from the effective group was significantly lower than that in the ineffective group (χ²=3.947, P=0.047). In the effective group, positive expression of HMGB1 in the para-carcinoma tissues was detected in 5 cases (8.8%) while there were 3 cases in the ineffective group (10.3%) and the difference was statistically significant (χ²<0.001, P=1.000) (Fig. 1). Survival time in the HMGB1-positive cases was obviously shorter than that in the negative cases (log-rank test χ²=65.384, P<0.001; Fig. 2).

In the effective group, the HMGB1 mRNA expression level was obviously lower than that in the ineffective group (0.16±0.02 vs. 0.44±0.03, t=8.246, P<0.001), and also, the protein expression level was lower in the effective group than that in the ineffective group; differences were statistically significant (P<0.05; Fig. 3).